Team:William and Mary/Improve

Improve

For William and Mary’s improved part we modified mScarlet-I to be compatible with our 3G assembly method. mScarlet-I is a rapidly-maturing version of mScarlet, a red fluorescent protein designed by Bindels et al(1). mScarlet-I differs from the original mScarlet protein by a single amino acid substitution T741.

We improved the mScarlet-I by flanking it with prefix sticky end C and suffix sticky end D in a 3G compatible backbone. These additions allow for the use of 3G mScarlet-I in 3G Assembly, a Gibson-Golden Gate hybrid assembly method. Specifically, 3G mScarlet-I's sticky ends correspond to the suffix sticky end C in all 3G RBS and the prefix sticky end D in all 3G terminators. This allows for great modularity in the Golden Gate reaction of 3G, as various 3G parts can be combined with 3G mScarlet-I to produce unique transcriptional units. 3G's modularity continues into the following Gibson reaction, where transcriptional units with corresponding UNS sequences can be "swapped out" to create unique circuits. This 3G compatibility makes 3G mScarlet-I more modular and more efficient for DNA assembly than the previous mScarlet-I.Please see BBa_K2680251 for more characterization showing is an improved version of BBa_K2333009

This gel extraction image displays two transcriptional units, both containing mScarlet-I. These transcriptional units allow us to perform 3G assembly; the left most transcriptional is composed of promoter R0051, 5’ UTR B0034, coding sequence mScarlet-I, and a Spy terminator. The right most transcriptional unit is made from promoter ptlpA, 5’ UTR B0034m, coding sequence mScarlet-I, and a thrL terminator.

Without the modifications from our sticky ends, these transcriptional units would not have been possible and it would have taken several days in assemble all the requisite parts needed for our circuit.

References

[1] Single Day Construction of Multigene Circuits with 3G Assembly. Andrew D. Halleran, Anandh Swaminathan, and Richard M. Murray, ACS Synthetic Biology 2018 7 (5), 1477-1480. DOI: 10.1021/acssynbio.8b00060